Compounds containing a vascular disrupting agent

ABSTRACT

The present invention relates to compounds, and pharmaceutically acceptable salts thereof, comprising a vascular disrupting agent (VDA) associated and a MMP proteolytic cleavage site. The compounds are useful in the treatment of cancer.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/148,368, filed on May 6, 2016, which is a continuation of U.S.application Ser. No. 14/109,333, filed on Dec. 17, 2013, now U.S. Pat.No. 9,358,303, which is a continuation of U.S. application Ser. No.12/595,482, now U.S. Pat. No. 8,691,751, which is a national stageapplication, filed under 35 U.S.C. § 371, of International Applicationno. PCT/GB2008/001043, filed on Mar. 27, 2008, which claims priority toGB Application No. 0707034.5, filed on Apr. 12, 2007. The contents ofeach of these applications are herein incorporated by reference in theirentireties.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The contents of the text file named “76317222.txt”, which was created onMar. 15, 2018 and is 2.99 KB in size, is hereby incorporated byreference in their entirety.

FIELD OF THE INVENTION

The present invention relates to compounds that are activated in vivofrom relatively biologically inactive compounds, so called “prodrugs”,to relatively biologically active compounds. The invention also relatesto the use of such compounds in the targeted treatment of cancer.

BACKGROUND TO THE INVENTION

The clinical utility of many anti-tumour compounds is restricted bytheir toxicity towards healthy cells, resulting in a narrow therapeuticindex and subsequent reduction in treatment benefit. Therefore, it wouldbe advantageous to target the drug selectively to the tumour andconsequently reduce normal tissue toxicity and side effects. One meansof approaching this objective is the design of prodrug molecules thatare specifically targeted to or selectively activated in tumour tissue,thereby reducing systemic levels of active drug and increasing thetherapeutic index.

Tumour growth, survival and dissemination of metastatic tumour cells isdependent on the presence of a functioning vascular network within thetumour (Tozer et al., Nat Rev Cancer, 5, 423-435 (2005)). Disruption ofeven a small proportion of the tumour vasculature has been demonstratedto induce wide ranging tumour death and retardation of metastasis, sincea single blood vessel is responsible for supporting the survival of manytumour cells. Endothelial cells, which form the major component of thevasculature, are highly dependent upon the tubulin cytoskeleton fortheir motility, invasion, attachment, alignment and proliferation(Denekamp, J, Br J Cancer, 45, 136-139 (1982)). Agents which disrupt theendothelial microtubule network will therefore cause a rapid collapse intumour blood flow and a prolonged period of vascular shutdown,culminating in extensive tumour-cell necrosis (Tozer et al., Nat RevCancer, 5, 423-435 (2005); Lippert J W, Bioorg Med Chem, 15, 605-615(2007)).

Colchicine and its analogues are potent vascular disrupting agents (VDA)causing haemorrhage and subsequent extensive necrosis in tumours (Tozeret al., Nat Rev Cancer, 5, 423-435 (2005)), as a direct consequence oftubulin binding and induction of microtubule depolymerisation (Chaudriet al., J MoI Biol, 303, 679-692 (2000)). Colchicine has not, however,shown intrinsic value as a clinically applicable anticancer therapeuticdue to a high level of toxicity and consequent very narrow therapeuticindex (Tozer et al., Nat Rev Cancer, 5, 423-435 (2005); Quinn et al., JMed Chem, 24, 636-639 (1981)). Major efforts in several laboratorieshave focused on development of small molecule VDAs with clinicalpotential, notably ZD6126 (phosphate analogue of N-acetylcolchinol) andcombretastatin-A4-phosphate (Lippert J W, Bioorg Med Chem, 15, 605-615(2007)), however, despite showing potency in preclinical evaluation andclinical trials, both cardiotoxicity and undesirable effects on thenormal vasculature are reported for these agents (Lippert J W, BioorgMed Chem, 15, 605-615 (2007); Beerepoot et al., J Clin Oncol, 24,1491-1498 (2006); Rustin et al., J Clin Oncol, 21, 2815-2822 (2003)).

The present inventors have developed a system for overcoming the toxiceffect of systemic administration of certain potent anti-cancer agentssuch as vascular disrupting agents.

STATEMENTS OF THE INVENTION

According to a first aspect of the invention there is provided acompound, or pharmaceutically acceptable salt thereof, comprising avascular disrupting agent (VDA) associated with a MMP proteolyticcleavage site. The term “associated with” in the context of theinvention is intended to include all direct and indirect means ofassociation, generally covalent, including, but not limited to, chemicalcross-linking or peptide bond linkage.

Pharmaceutically-acceptable salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulfuric,nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic,succinic, and the like. Also, pharmaceutically-acceptable salts can beprepared as alkaline metal or alkaline earth salts, such as sodium,potassium or calcium salts.

In a preferred aspect the invention provides a compound of formula (I)X−Y  (I)whereinX is a vascular disrupting agent (VDA);Y is a matrix metalloproteinase (MMP) proteolytic cleavage site.

Compounds according to the invention provide prodrugs which areconverted to an active and potent VDA within the tumour environment byoverexpressed MMPs. Tumour selective activation of a prodrug of theinvention increases tumour levels, and decreases systemic levels, of theVDA and optionally additional active ingredients thereby greatlyincreasing their therapeutic index and efficacy.

VDAs comprise a multi-ring system, for example a fused or unfusedbicyclic or tricyclic ring system. Thus X includes any multi-ring systemof a VDA that is capable of binding to and disrupting tumour bloodvessels.

VDAs are compounds which directly interact with tubulin and consequentlyintracellular microtubules, resulting in a cytostatic or cytotoxiceffect. VDAs can be divided into three classes:

-   -   (i) those compounds that interact with tubulin at the colchicine        binding site on tubulin;    -   (ii) those compounds that share a common binding site on tubulin        with the Catharanthus (Vinca) alkaloids;    -   (iii) compounds that promote the formation of stable        microtubules in a manner similar to paclitaxel, a novel taxane        diterpenoid isolated from the bark of the Pacific yew.

Thus in a preferred aspect of the invention the VDA is a tubulin bindingagent. The tubulin binding agent may be selected from the groupconsisting of i) those which interact with tubulin at the colchicinebinding site: including but not limited to colchicine (includingcolchicine analogues such as N-acetylcolchinol-O-phosphate (ZD6126) andABT-751), colchicinoids, combretastatins, phenstatin, podophyllotoxins,steganacins, amphethinile and stilbenes, and ii) those which interactwith the Vinca binding site of tubulin: including but not limited tovincristine, vinblastine, vinflunine, maytansinoids, phomopson A,rhizoxin, auristatin (including analogues thereof) and dolastatin

In a yet preferred aspect of the invention the VDA is a tubulin bindingagent that interacts with the colchicine binding site within tubulin.

Colchicine derivatives may include, but are not limited to,azademethylcolchicine, azacolchicine, N-methyl desacetylcolchicine,desacetylcolchicine.

The VDA may include tubulin binding agents including combretastatins(e.g. combretastatin A-4 3-0-phosphate), auristatin (including analoguesthereof), dolastatin; and flavenoids (e.g. tumour necrosis factor α and5,6-dimethylxanthenone-4-acetic acid (DMXAA), flavone acetic acid(FAA)). Thus in an alternative embodiment of the invention the VDA is acombretastatin.

The invention includes any member of the MMP family Proteolytic cleavageat the MMP cleavage site by an MMP releases the VDA, and any otheractive agent associated with the MMP cleavage site, in active form.

The MMP family is divided into eight structural groups: five of whichare secreted and three which are membrane-type MMPs (MT-MMPs). MT-MMPsare localised on the cell surface. The invention includes secreted MMPsand membrane type MMPs.

In a preferred aspect of the invention the MMP is a membrane-type(MT-MMP). The MT-MMP may be selected from the group consisting of

-   -   (i) type I transmembrane type MT-MMPs for example MMP-14        (MT1-MMP), MMP-15 (MT2-MMP), MMP-16 (MT3-MMP) and MMP-24        (MT5-MMP);    -   (ii) glycosyl phosphatidylinositol (GPI)-anchored structural        group of MT-MMPs for example MMP-17 (MT4-MMP) and MMP-25        (MT6-MMP);    -   (iii) type I1 transmembrane class for example MMP-23. The        invention also encompasses any MMP proteases subsequently        identified and classified into one of these MT-MMP subfamilies.

The MMP cleavage site may comprise any peptide sequence having an amidebond, typically a peptide bond, that is cleavable by a MMP. Preferably Yis a peptide sequence comprising between two and twenty amino acids, forexample between four and ten amino acids (e.g. 7 or 8 amino acids). Theamino acids of the peptide sequence may include any naturally occurringor synthetic D- or L-amino acid, preferably L- or combinations of D- andL-amino acids.

The invention also includes peptide analogues, for example peptidemimics, of the peptide sequence in which for example an amide bond isreplaced with olefinic bonds, Nα- and Cα-methylated amino acids,unnatural amino acids and other approaches known in the art. Suchpeptidomimetic approaches are used in the art to enhance the specificityof cleavage thereby serving to diminish undesired enzymatic hydrolysis.

In a preferred embodiment of the invention the MMP proteolytic cleavagesite comprises a sequence of amino acids (e.g. 6 to 10 amino acids) inwhich one or more amino acids in the sequence namely serine, threonineand/or tyrosine are glycosylated to enhance hydrophilicity and as suchsolubility. O-glycosylation of suitable amino acids in the sequencenamely serine, threonine and tyrosine may enhance hydrophilicity and assuch solubility. Preferably the MMP proteolytic cleavage site comprises8 amino acids.

The MMP proteolytic cleavage site may comprise the sequence

-   P4-P3-P2-P1-P1′-P2′-P3′-P4′    wherein P1 to P4 and P1′ to P4′, which may be the same or different,    are amino acid residues and wherein proteolytic cleavage takes place    between residues P1 and P1′. Preferably P1 and P1′ are different.    Preferably still P1 to P4 are different. Preferably P1′ to P4′ are    different.

In one embodiment P1 is GIy and/or P1′ is Hof.

The MMP proteolytic cleavage site may comprise the amino acid sequence(i)

-Cit-Gly-Hof-Tyr-Leu- (SEQ ID NO: 1)

In an embodiment of the invention the MMP proteolytic cleavage sitecomprises the amino acid sequence (ii)

-K-K-Cit-Gly-Hof-Tyr-Leu- (SEQ ID NO: 2)wherein K represents an amino acid which may be selected from the groupconsisting of, but not limited to, Cit, Gly, Glu and Pro.

In an embodiment of the invention the MMP proteolytic cleavage sitecomprises the amino acid sequence (iii)

-Glu-Pro-Cit-Gly-Hof-Tyr-Leu- (SEQ ID NO: 3)

In a preferred embodiment the MMP proteolytic cleavage site comprisesthe amino acid sequence (iv)

-Arg-Ser-Cit-Gly-Hof-Tyr-Leu- (SEQ ID NO: 4)

An embodiment the invention comprises a compound of formula (I) whereinY comprises a C-terminal site and an N-terminal site and wherein saidC-terminal site is directly or indirectly linked to X and saidN-terminal site is directly or indirectly linked to a further moiety forexample c or Z as described hereinbelow.

An alternative embodiment of the invention comprises a compound offormula (I) wherein Y comprises a C-terminal site and an N-terminal siteand wherein said N-terminal site is directly or indirectly linked to Xand said C-terminal site is directly or indirectly linked to a furthermoiety for example c or Z as described hereinbelow.

In one preferred embodiment the invention there is provided a compoundof formula (I) wherein X is colchicine or an analogue thereof and Y is apeptide comprising the amino acid sequence -Arg-Ser-Cit-Gly-Hof-Tyr-Leu-(SEQ ID NO: 4).

In a preferred aspect the invention provides a compound of formula (II)X−Y−c  (II)wherein X and Y are as defined herein;

c is an end group or “capping group”. Capping groups may be used to capa peptide chain in pharmaceutical use in order to prevent non-specificdegradation of the peptide for example by enzymes other than MMPs. c mayinclude any appropriate moiety on the N- or C-terminus acting as ablocking group including, but not limited to simple sugars, D-aminoacids, proline imino acids or fluorescein derivatives.

The present invention may further provide a “linker”. The linker may beprovided at the C and/or N terminus of Y. Preferably the linker isprovided at the C terminus of Y. Preferably the linker is continuouswith the amino acid sequence of Y. The linker may include any moietythat is associated with Y and which may be removed chemically,enzymatically or decompose spontaneously. The linker may consist of asingle amino acid (e.g. tyrosine) or may comprise an amino acidsequence. Where the linker comprises a sequence of amino acids, thesequence may provide a hydrophilic region that may facilitate cleavageby an MMP at Y. O-glycosylation of suitable amino acids in the sequencenamely serine, threonine and tyrosine may enhance hydrophilicity and assuch solubility.

Thus in a preferred aspect of the invention there is provided a compoundof formula (III)X−a−Y  (III)wherein X and Y are as defined herein; anda is a linker wherein the linker is directly or indirectly associatedwith X.

In an embodiment the invention provides a compound of formula (IV)X−a−Y−c  (IV)wherein X, a, Y and c are as defined herein.

In a yet further preferred aspect of the invention there is provided a“spacer” which may be the same as, or different to, the linker describedherein. The spacer may be provided at the C and/or N terminal of Y.Preferably the spacer is provided at the N terminus of Y and serves toprevent unwanted removal of c during synthesis. The spacer may bedirectly or indirectly associated with Y. The spacer may include anysingle amino acid (e.g. β-alanine), amino acid sequence, a succinylgroup. Thus the invention preferably provides a compound of formula (V)X−Y−b−c  (V)wherein X, Y, and c are as defined herein;b is a spacer as defined herein.

In a further embodiment the invention provides a compound of formula(VI)X−a−Y−b−c  (V1)wherein X, Y, a, b and c are as defined herein.

In one embodiment of the invention there is provided a compound offormula (VI) wherein X is colchicine (or an analogue thereof), Y is apeptide comprising the amino acid sequence -Arg-Ser-Cit-Gly-Hof-Tyr-Leu-(SEQ ID NO: 4), a is tyrosine, b is alanine and c is fluorescein or aderivative thereof.

In a second aspect of the invention there is provided a compound, orpharmaceutically acceptable salt thereof, of formula (VII)X−Y−Z  (VII)wherein X and Y are as defined herein; Z is an anti-cancer agent.

Preferably Z is an anticancer agent selected from the group consistingof a vascular disrupting agent (preferably different to X), anantimetabolite (e.g. 5-fluorouracil), a cytotoxic or antiproliferativeagent (e.g. anthracycline (e.g. doxorubicin), vinca alkaloid, taxane,cytotoxic nucleotide), a biotoxin, radiotherapeutic, hormonal agent orany natural products or agents known to induce a cytotoxic, cytostatic,anti-angiogenic or vascular disrupting effect.

In a preferred aspect of the invention there is provided a compound offormula (VIII)X−a−Y−Z  (VIII)wherein X, a, Y and Z are as defined herein.

In a yet preferred aspect of the invention there is provided a compoundof formula (IX)X−a−Y−b−Z  (IX)wherein X, a, Y, b and Z are as defined herein. In this aspect of theinvention, the purpose of the spacer b is to convert the N-terminalamine of Y into a carboxylic acid to allow attachment of a compound Zwherein Z bears a free amine (for example where Z is doxorubicin). WhereZ bears a free carboxylic acid, b is not required.

In one preferred embodiment the invention there is provided a compoundof formula (VII) wherein X is colchicine (or an analogue thereof) and Zis doxorubicin. Preferably still Y is a peptide comprising the aminoacid sequence -Arg-Ser-Cit-Gly-Hof-Tyr-Leu- (SEQ ID NO: 4).

The extent of protection includes counterfeit or fraudulent productswhich contain or purport to contain a compound of the inventionirrespective of whether they do in fact contain such a compound andirrespective of whether any such compound is contained in atherapeutically effective amount.

Included in the scope of protection are packages which include adescription or instructions which indicate that the package contains aspecies or pharmaceutical formulation of the invention and a productwhich is or comprises, or purports to be or comprise, such a formulationor species. Such packages may be, but are not necessarily, counterfeitor fraudulent.

A further aspect of the invention provides a process for preparing acompound according to the invention the process comprising the steps of

-   i) providing a solid support attached to X;-   ii) optionally attaching a linker a to the C or N terminal of X;-   iii) attaching amino acid residues step-wise to the C or N terminal    of X, or the linker attached to X in (ii), to provide the peptide    sequence Y containing the MMP proteolytic cleavage sequence;-   iii) optionally attaching a capping group c to the respective C or N    terminal of Y to provide a compound of formula (II) or (IV).

In a preferred process the solid support is any polymeric support suchas any polystyrene based or PEG based resin for example trityl-basedresins.

In a further aspect the invention provides the use of a MMP proteolyticcleavage site in the site specific activation of a VDA. The term “sitespecific activation” as used herein means, in general terms and notlimited to, the activation of a VDA by site specific cleavage at the MMPproteolytic cleavage site. Site specific cleavage at the proteolyticcleavage site is expected to take place concomitantly with the releaseand hence activation of the VDA.

Pharmaceutical Compositions and Uses

In other aspects the invention provides a compound, or pharmaceuticallyacceptable salt thereof, as hereinbefore described for use in medicine.In further aspects, there is provided a pharmaceutical formulationcomprising a compound as hereinbefore described. The formulation maycontain at least one additional pharmaceutically acceptable componente.g. an excipient, diluent or carrier. Preferably the formulation isintended for parenteral administration.

The invention provides a pharmaceutical formulation comprising acompound according to the invention. In a preferred embodiment, thecompound is of formula (VII).

In a preferred aspect of the invention said composition includes apharmaceutically acceptable carrier or diluent.

The compositions of the invention are typically administered ineffective amounts. An “effective amount” is that amount of a compositionthat alone, or together with further doses, produces the desiredresponse. When administered, the pharmaceutical compositions of thepresent invention are administered in pharmaceutically acceptablepreparations. Such preparations may routinely contain pharmaceuticallyacceptable concentrations of salt, buffering agents, preservatives,compatible carriers and optionally other therapeutic agents (forexample, cisplatin; carboplatin; cyclosphosphamide; melphalan;carmusline; methotrexate; 5-fluorouracil; cytarabine; mercaptopurine;daunorubicin; doxorubicin; epirubicin; vinblastine; vincristine;dactinomycin; mitomycin C; taxol; L-asparaginase; G-CSF; etoposide;colchicine; derferoxamine mesylate; and camptothecin).

The compositions of the invention can be administered by anyconventional route, including injection or by gradual infusion overtime. The administration may, for example, be oral, intravenous,intraperitoneal, intramuscular, intracavity, subcutaneous, ortransdermal. In the case of treating a particular disease, such ascancer, the desired response is inhibiting the progression of thedisease. This may involve only slowing the progression of the diseasetemporarily, although more preferably, it involves halting theprogression of the disease permanently. This can be monitored by routinemethods known in the art.

Administration of the compositions to mammals other than humans, (e.g.for testing purposes or veterinary therapeutic purposes), is carried outunder substantially the same conditions as described above. A subject,as used herein, is a mammal, preferably a human, and including anon-human primate, cow, horse, pig, sheep, goat, dog, cat or rodent.

When administered, the pharmaceutical preparations of the invention areapplied in pharmaceutically-acceptable amounts and inpharmaceutically-acceptable compositions. The term “pharmaceuticallyacceptable” means a non-toxic material that does not interfere with theeffectiveness of the biological activity of the active ingredients.

Pharmaceutical compositions may be combined, if desired, with apharmaceutically-acceptable carrier. The term“pharmaceutically-acceptable carrier” as used herein means one or morecompatible solid or liquid fillers, diluents or encapsulating substanceswhich are suitable for administration into a human. The term “carrier”denotes an organic or inorganic ingredient, natural or synthetic, withwhich the active ingredient is combined to facilitate the application.

The pharmaceutical compositions may contain suitable buffering agents,including: acetic acid in a salt; citric acid in a salt; boric acid in asalt; and phosphoric acid in a salt. The pharmaceutical compositionsalso may contain, optionally, suitable preservatives, such as:benzalkonium chloride; chlorobutanol; parabens and thimerosal.Compositions suitable for oral administration may be presented asdiscrete units, such as capsules, tablets, lozenges, each containing apredetermined amount of the active compound. Other compositions includesuspensions in aqueous liquids or non-aqueous liquids such as syrup,elixir or an emulsion.

Compositions suitable for parenteral administration convenientlycomprise a sterile aqueous or non-aqueous preparation of compound, whichis preferably isotonic with the blood of the recipient. This preparationmay be formulated according to known methods using suitable dispersingor wetting agents and suspending agents. The sterile injectablepreparation also may be a sterile injectable solution or suspension in anontoxic parenterally-acceptable diluent or solvent, for example, as asolution in 1,3-butane diol. Among the acceptable vehicles and solventsthat may be employed are water, Ringer's solution, and isotonic sodiumchloride solution. In addition, sterile, fixed oils are conventionallyemployed as a solvent or suspending medium. For this purpose any blandfixed oil may be employed including synthetic mono- or di-glycerides. Inaddition, fatty acids such as oleic acid may be used in the preparationof injectables. Carrier formulation suitable for oral, subcutaneous,intravenous, intramuscular, etc. administrations can be found inRemington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.

In an aspect of the invention, the compounds according to the inventionmay be used to treat a disease or condition associated with tissueexpressing an MMP, particularly a cancer.

The invention provides a method to treat cancer in a subject comprisingadministering an effective amount of a compound according to theinvention. In a preferred method of the invention said subject is human.

As used herein, the term ‘cancer’ refers to cells possessing thecapacity for autonomous growth i.e., an abnormal state or conditioncharacterised by rapidly proliferating cell growth. The term is meant toinclude all types of cancerous growths or oncogenic processes,metastatic tissues or malignantly transformed cells, tissues, or organs,irrespective of histopathological type or stage of invasiveness. Theterm ‘cancer’ includes malignancies of epithelial, endodermal andmesenchymal origin, particularly carcinomas and sarcomas, such as thoseaffecting the respiratory system (mouth, nose, trachea, lung),gastrointestinal tract (tongue, esophagus, stomach, small intestines,colon, liver, pancreas, gall bladder, rectum), endocrine system(thyroid, pituitary, adrenal glands), genito-urinary tract (urinarybladder, kidney), reproductive system (breast, ovaries, uterus, cervix,prostate, penis, scrotum, testes), skin (melanocytes, epidermis,endodermis), nervous system (brain, spinal cord, glial cells, neurons)and lymphoid system.

The term ‘carcinoma’ is art recognised and refers to malignancies ofepithelial origin including respiratory system carcinomas,gastrointestinal system carcinomas, endocrine system carcinomas,genito-urinary tract carcinomas, skin carcinomas, and carcinomas of thereproductive system. The term “carcinoma” also includes“adenocarcinomas” referring to carcinomas deriving from glandulartissue, “squamous carcinomas” referring to carcinomas of squamousorigin, and “carcinosarcomas” referring to tumours composed ofcarcinomatous and sarcomatous tissue. Exemplary carcinomas include thoseforming from the epithelia of the cervix, prostate, breast, nose, headand neck, oral cavity, esophagus, stomach, liver, pancreas, colon,ovary, urinary bladder and lung, particularly non-small lung carcinoma.

The term ‘sarcoma’ is art recognised and refers to malignancies of softtissues or connective or supportive tissue, including bone, cartilage,adipose tissue, smooth muscle, skeletal muscle, nerve sheath, bloodvessels, mesothelium, and gastrointestinal stroma. Further types ofcancer include “leukaemias” which refer to tumours deriving from whiteblood cells, and “lymphomas” referring to tumours of the lymphoidsystem.

A pharmaceutical formulation comprising a compound according to theinvention may be administered in combination, either sequentially or ata substantially similar time, as an anti-cancer agent including, but notlimited to, an antimetabolite (e.g. 5-fluorouracil), a cytotoxic oranti-proliferative agent (e.g. anthracycline, vinca alkaloid, taxane,cytotoxic nucleotide), a biotoxin, radiotherapeutic, hormonal agent orany natural products or agents known to induce a cytotoxic, cytostatic,anti-angiogenic or vascular disrupting effect.

As used herein, “treatment” refers to clinical intervention in anattempt to alter the natural course of the individual or cell beingtreated, and may be performed either for prophylaxis or during thecourse of clinical pathology.

In a further aspect the invention provides the use of a compoundaccording to the invention in the manufacture of a medicament to treatcancer.

In one aspect of the present invention the compounds or compositions ofthe invention may be used to treat an inflammatory disorder and/or aninflammatory response. Thus, according to a further of the inventionthere is provided a method to treat an inflammatory disorder in asubject comprising administering an effective amount of a compoundaccording to the invention.

The inflammatory disorder may be selected from the group consisting ofconsisting of atherosclerosis, rheumatoid arthritis, osteoarthritis,gout, lupus erythematosus, scleroderma, Sjorgen's syndrome, poly- anddermatomyositis, vasculitis, tendonitis, synovitis, bacterialendocarditis, periodontitis, osteomyelitis, psoriasis, pneumonia,fibrosing alveolitis, chronic bronchitis, bronchiectasis, emphysema,silicosis, pneumoconiosis, tuberculosis, ulcerative colitis, Crohn'sdisease, chronic inflammatory demyelinating polyradiculoneuropathy,chronic inflammatory demyelinating polyneuropathy, multiple sclerosis,Guillan-Barre Syndrome and myasthemia gravis, mastitis, laminitis,laryngitis, chronic cholecystitis, Hashimoto's thyroiditis, andinflammatory breast disease. In one embodiment, the inflammatorydisorder may be the result of tissue or organ rejection aftertransplantation. In particular embodiments, the inflammatory disorder isselected from the group consisting of atherosclerosis, rheumatoidarthritis, osteoarthritis, sepsis and polyarthritis.

The compounds of the invention may be used to treat heart failure. Alsoprovided is a use of a compound as described herein for the manufactureof a medicament to treat heart failure.

In one embodiment of the present invention the compounds of theinvention may be useful in treating a wound (e.g. ulcers, lesionsincluding cutaneous cuts or burns). Thus the invention provides a methodto treat a wound in a subject comprising administering an effectiveamount of a compound according to the invention. In a preferred methodof the invention said subject is human.

The compounds of the invention may also be used to treat conditions anddisorders associated with menstruation.

There is further provided a package or kit of parts comprising:

-   (1) a compound or composition described herein; together with-   (2) instructions to use the compound in a method or use described    herein.

The package defined herein may comprise more than one dosage unit inorder to provide for repeat dosing. If more than one dosage unit ispresent, such units may be the same, or may be different in terms of thedose of the compound composition and/or physical form.

Throughout the description and claims of this specification, the words“comprise” and “contain” and variations of the words, for example“comprising” and “comprises”, means “including but not limited to”, andis not intended to (and does not) exclude other moieties, additives,components, integers or steps.

Throughout the description and claims of this specification, thesingular encompasses the plural unless the context otherwise requires.In particular, where the indefinite article is used, the specificationis to be understood as contemplating plurality as well as singularity,unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties orgroups described 5 in conjunction with a particular aspect, embodimentor example of the invention are to be understood to be applicable to anyother aspect, embodiment or example described herein unless incompatibletherewith.

The invention will now be described by way of example only withreference to the 0 following Figures in which:

FIG. 1 is a graph showing the metabolism of prodrug-1 versus time intumour and non-tumour tissues ex vivo;

FIG. 2 is a graph showing accumulation and levels of prodrug-1 intumour-bearing mice following intraperitoneal administration;

FIG. 3 is a graph demonstrating levels of VDA accumulating followingintraperitoneal administration of prodrug-1 to tumour bearing mice;

FIG. 4 is a graph showing differential metabolism of prodrug-2 in tumourhomogenates expressing high MT1-MMP levels (HT1080) versus tumourhomogenates expressing low MT1-MMP levels (MCF-7);

FIG. 5 is a graph showing accumulation and levels of prodrug-2 in HT1080tumour bearing mice following intraperitoneal administration;

FIG. 6 is a graph demonstrating levels of VDA accumulating followingintraperitoneal administration of prodrug-2 to HT1080 tumour bearingmice;

FIG. 7 is a schematic of MMP-activated prodrug (compound i) targetingall MMPs showing the amino acid sequence of SEQ ID NO: 6.Hof=homophenylalanine; Cit=citrulline;

FIG. 8 is a schematic of MMP-activated prodrug (compound i) targetingonly Membrane-Type WIMPs (MT-MMTPs) showing the amino acid sequence ofSEQ ID NO: 7.

EXAMPLE Materials and Methods Synthesis of Immobilised ColchicineDerivative

Synthesis of 1

Ammonia solution (35%, 15 mL) was added to colchicine (750 mg, 1.88mmol, 1.00 eq) and the reaction mixture stirred at room temperatureovernight. The crude product was washed with KHSO₄ (1 M, aq), dried withMgSO₄, filtered and concentrated under reduced pressure. It wassubsequently purified by flash chromatography on silica gel (gradientelution: CH₂Cl₂/methanol 95:5 to 10:1) to give 1 as a yellow solid (427mg, 1.11 mmol, 59%). δ_(H) (600 MHz, CDCI₃), 7.99 (1H, broad s, NH),7.56 (1H, d, J 2.1, C8-H), 7.32 (1H, d, J 10.7, C11-H), 6.88 (1H, d, J11.0, C10-H), 6.52 (1H, s, C4-H), 6.03 (2H, broad s, NH₂), 4.68 (1H,ddd, J 12.6, 6.5 and 6.5, C7-H), 3.93 (3H, s, OCH₃), 3.88 (3H, s, OCH₃),3.60 (3H, s, OCH₃), 2.47 (1H, dd, J 13.4 and 6.2, C5-CH₂), 2.35 (1H,ddd, J 13.4, 12.7 and 6.9, C5-CH₂), 2.29-2.23 (1H, m, C6-CH₂), 1.98 (3H,s, CH₃), 1.90-1.88 (1H, m, C6-CH₂); ES m/z (%) 385 [M⁺+H](100).

Synthesis of 2

HCTU (642 mg, 1.55 mmol, 1.50 eq) and diisopropylethylamine (DiPEA, 516μL, 404 mg, 3.11 mmol, 3.00 eq) were added to a solution ofFmoc-tyr(tBu)-OH (714 mg, 1.55 mmol, 1.50 eq) in DMF (10 mL). Afterstirring at room temperature for 5 minutes, 1 (398 mg, 1.04 mmol, 1.00eq) was added to the solution. The reaction mixture was stirred at 50°C. for 22 h. DMF was removed in vacuo and the resultant oil wasdissolved in CH₂CI₂ (20 mL). The organic phase was washed with KHSO₄(aq, 2×20 mL), dried with MgSO₄ and concentrated under reduced pressure.The crude product was purified by flash chromatography (gradientelution: CH₂Cl₂/methanol 100:0 to 99:1 to 98:2) to give 2 as a yellowsolid (530 mg, 642 mol, 67%).

δ_(H) (600 MHz, CDCI₃), 10.42 (1H, broad s, NH), 9.02 (1H, d J 10.7,C11-H), 7.75 (2H, d, J 7.2, C23-H, C24-H), 7.54 (2H, d, J 7.2, C20-H,C27-H), 7.45 (1H, d, J 11.0, C10-H), 7.39 (2H, dd, J 7.2 and 7.2, C22-H,C25-H), 7.29 (2H, dd, J 6.6 and 6.6, C21-H, C26-H), 7.19 (1H, broad s,C8-H), 7.03 (2H, d, J 7.9, C14-H, C17-H), 6.81 (2H, d, J 7.9, C15-H,C16-H), 6.50 (1H, s, C4-H), 5.88 (1H, broad s, NH), 5.25 (1H, broad s,C12-H), 4.73-4.67 (1H, m, C7-H), 4.43 (1H, dd, J 10.0 and 7.6, C18-CH₂),4.28 (1H, dd, J 10.0 and 7.2, C18-CH₂), 4.16 (1H, dd, J 7.2 and 6.19,C19-H), 3.93 (3H, s, OCH3), 3.88 (3H, s, OCH₃), 3.62 (3H, s, OCH₃), 3.21(1H, dd, J 13.1 and 4.8, C13-CH₂), 3.11 (1H, dd, J 13.1 and 5.5,C13-CH₂), 2.53 (1H, dd, J 13.4 and 6.2, C5-CH₂), 2.40 (1H, ddd, J 13.4,12.7 and 6.9, C5-CH₂), 2.22-2.15 (1H, m, C6-CH₂), 1.88 (3H, s, CH₃),1.80 (1H, ddd, J 11.5, 11.3 and 6.9, C6-CH₂), 1.22 (9H, s, C(CH₃)₃); ESm/z (%) 826 [M⁺](100).

Synthesis of 3

TFA (2 mL) was added to a solution of 2 (486 mg, 589 μmol, 1.00 eq) andthe reaction mixture stirred for 20 min. TLC indicated quantitativeconversion to the product. The product was concentrated under reducedpressure, with toluene co-evaporation to give 3 in quantitative yield.

δ_(N)(600 MHz, CDCI₃), 10.08 (1H, broad s, NH), 8.99 (1H, d J 10.7,C11-H), 7.71 (2H, d, J 6.2, C23-H, C24-H), 7.55 (1H, s, C8-H), 7.49 (2H,dd, J 6.5 and 6.5, C20-H, C27-H), 7.41 (1H, d, J 10.2, C10-H), 7.33 (2H,dd, J 6.2 and 6.2, C22-H, C25-H), 7.26-7.21 (2H, m, C21-H, C26-H), 6.91(2H, d, J 8.3, C14-H, C17-H), 6.56 (2H, d, J 7.2, C15-H, C16-H), 6.45(1H, S, C4-H), 5.93 (1H, broad s, NH), 5.28 (1H, s, NH), 4.95-4.90 (1H,m, C12-H), 4.60 (1H, ddd, J 11.7, 5.8 and 6.9, C7-H), 4.39 (1H, dd, J8.9 and 8.6, C18-CH₂), 4.29-4.24 (1H, m, C18-CH₂), 4.12 (1H, dd, J 6.9and 6.9, C19-H), 3.90 (3H, s, OCH₃), 3.84 (3H, s, OCH₃), 3.54 (3H, s,OCH₃), 3.08 (2H, d, J 5.2, C13-CH₂), 2.44 (1H1 dd, J 13.4 and 6.2,C5-CH₂), 2.33-2.26 (1H, m, C5-CH₂), 2.15-2.09 (1H, m, C6-CH₂), 1.82 (3H,s, CH₃), 1.75-1.69 (1H, m, C6-CH₂); ES m/z (%) 770 [M⁺](100).

Preparation of 4:

2-Chlorotrityl chloride resin (Novabiochem, 100-200 mesh, substitution1.4 mmolg⁻¹, 589 mg, 0.765 mmol, 1.00 eq) was suspended in a solution of3 (589 mg, 0.765 mmol, 1.00 eq), dimethylaminopyridine (10 mg, 76.5μmol, 0.01 eq), DiPEA (247 mg, 1.913 mmol, 333 μL, 2.50 eq) and pyridine(241 mg, 3.061 mmol, 248 μL, 4.00 eq) in THF (10 mL) and stirred for 6hours at 50° C. The resin was subsequently filtered and washedthoroughly with THF. The resin was then capped by washing the resincarefully with methanol (CH₂CI₂:MeOH:DiPEA 17:2:1, 100 mL). Resin 4 wasdried overnight over P2O5. Dry resin weight: 593 mg (loading 56%).

General Procedure for Synthesis of Endopeptidase-Activated Pro-Drugs

As an example, peptide conjugate 5 was synthesised using conventionalsolid phase peptide synthesis, from immobilised colchicine derivative 4,using an Fmoc-based strategy.

N^(α)-Fmoc strategy synthesis of peptide acids was achieved manuallyusing 2-chlorotrityl-derivatised resin 4. The resin was swelledthoroughly in DMF, followed by removal of the N-Fmoc protecting group bytreatment with 20% v/v piperidine in DMF (3×3 min). All couplings wereperformed in DMF, employing 2.5-fold molar excesses of N^(α)-Fmocprotected amino acids (with appropriate side-chain protecting groups),and activated using HCTU/HOBt/DiPEA. N^(α)-Fmoc deprotections wereperformed using 20% piperidine in DMF (3×3 min). The success ofcouplings and de-protections was monitored using the ninhydrin-basedKaiser test. Unsuccessful couplings were repeated. After the finalN^(α)-Fmoc deprotection, the peptide chain was endcapped withfluorescein isothiocyanate (2.50 eq, in the presence of DiPEA1 1.50 eq).The success of this reaction was also monitored by the Kaiser test.

An additional β-alanine residue was incorporated into the sequence toovercome incompatability of the thiourea linkage and the acidicconditions of cleavage (the thiourea can rearrange, and the carbonylcarbon of the preceding amide bond can undergo nucleophilic attack bythe sulphydryl-like function so formed. This leads to cleavage of theamide bond, with concomitant formation of a cyclic thiazolinone. Thethiazolinone can undergo rearrangement in the presence of aqueous acidto form a thiohydantoin).

On completion of the sequence, the resin was washed (DMF, CH2CI2,CH2CI2/MeOH) and dried in vacuo over KOH to constant weight. Peptideswere cleaved from the resin by mild acidolysis usingTFA-H20-triisopropylsilane 95:2.5:2.5 for 2 h at RT, with simultaneousside-chain de-protection. Following cleavage, the TFA was removed underreduced pressure. The crude product was extracted into 95% aqueousacetic acid and lyophilised. The crude peptide was subsequently analysedusing reversed phase HPLC and purified using preparative HPLC(purity>97%). Pure fractions were combined and lyophilised. Identity wasconfirmed by mass spectrometry.

Potential Attachment of Parent Colchicine to a Peptide Sequence Throughthe B-Ring

To enable attachment of a colchicine moiety through the peptideN-terminus, the following strategy will be used. The B-ring amine can bede-masked using published methods. Acylation with aspartic acid willintroduce a carboxylic acid to the molecule (from the amino acid sidechain) thereby enabling conjugation to the free amine at the peptideN-terminus (see below).

Acetylation of the amide bond will also be examined, to assess whetherparent colchicine is released following MMP activation and subsequentexopeptidase degradation.

Strategies for the Attachment of Doxorubicin to a Peptide Sequence

To enable attachment of doxorubicin through the peptide N-terminus(following peptide synthesis using the immobilised colchicinederivatised resin previously described) it must first be modified tointroduce a carboxylic acid. Examples include reaction with succinicanhydride (strategy 1, below). However, by utilising the side chain ofaspartic acid (both natural amino acids), as shown below (strategy 2) anatural amino acid (as opposed to a foreign chemical entity) is releasedon metabolism:

Strategy 1: (Succinyl Spacer)

Strategy 2: (Amino Acid Spacer)

C-Terminal Doxorubicin Linkage

Protection of the amine group of doxorubicin with Dde (a commonly usedprotecting group in peptide chemistry and by our group) would allowimmobilisation of the agent onto a trityl-based (or otherwise) resin.Subsequent removal of the Dde group would de-mask the amine, allowing apeptide sequence to be constructed from this point (i.e. through theC-terminus). Standard Fmoc-based solid phase synthesis would produce apeptide sequence. An appropriately derivatised VDA could then beconjugated through the N-terminus. Resin cleavage and purification wouldbe as previously described.

Incorporation of Glycosylated Amino Acids (to Enhance PhysicochemicalProperties)

Amino acids with appropriate side chain functionality (e.g. serine,tyrosine, threonine) can be glycosylated (with mono-, di- ortrisaccharides) to produce peptides with enhanced aqueous solubility.Such a carbohydrate-derivatised moiety could be used in place of serine,for example (see scheme below).

Results1) MMP-activated prodrug (compound i)

-   -   pan MMP targeted        Structure:

-   -   a) Prodrug-1 has been screened using normal mouse plasma, normal        mouse liver homogenate and experimental human tumour model        homogenate (HT1080 xenograft; known to express majority of MMPs)        ex vivo. Prodrug cleavage and metabolism were detected using        LC/MS/MS.        -   a. Prodrug-1 was stable in plasma and liver, supporting            systemic stability of these therapeutics (FIG. 1).        -   b. Prodrug-1 was metabolised in tumour homogenate,            supporting activation of these therapeutics in tumours            expressing MMPs (FIG. 1).    -   b) Prodrug-1 is cleaved rapidly at the Glycine-Homophenylalanine        (Gly-Hof) by recombinant MMP-2, MMP-9, MMP-10 and MMP-14 at        least. Demonstrated by LC/MS/MS and mass spectrometry (data not        shown)    -   c) Prodrug-1 was administered in vivo via the intraperitoneal        route to mice bearing subcutaneous HT1080 tumour model        (expression of majority of MMPs). Plasma, tissues and tumours        were collected at regular intervals post-dosing. Levels of        prodrug and VDA2 were assessed by LC/MS/MS.        -   a. Prodrug-1 accumulated and was detected in all tissues            analysed (FIG. 2).        -   b. Highest prodrug-1 levels were observed in the tumour            (FIG. 2).        -   c. Prodrug-1 not detectable after 24 hours post-dosing.            (FIG. 2)        -   d. VDA2 was detectable at low levels in normal tissues            following prodrug-1 administration (FIG. 3)        -   e. VDA2 levels were detected at high levels in tumour tissue            following prodrug-1 administration (FIG. 3)        -   f. VDA2 was still detectable at high levels in tumour and            was undetectable in normal tissues after 24 hours            post-dosing with prodrug-1 (FIG. 3)            2) MMP-activated prodrug (compound i)    -   targeted to Membrane-type WIMPs (MT-MMPs)        Structure:

-   -   d) Compound 1 was modified in order to change the MMP-targeting        of the compound from being pan-MMP to MT-MMP selective        (Prodrug-2)        -   a. Arginine was incorporated in place of the Glutamic acid            at the P4 position        -   b. Proline was removed and replaced with Serine at the P3            position    -   e) Prodrug-2 has been screened using normal mouse plasma, normal        mouse liver homogenate and experimental human tumour model        homogenates demonstrating high MT1-MMP (MMP-14) expression and        activity (HT1080) and low MT1-MMP expression and activity        (MCF-7) ex vivo. Prodrug-2 cleavage and metabolism were detected        using LC/MS/MS.        -   a. Prodrug-2 remained intact in plasma supporting systemic            stability of this therapeutic.        -   b. Prodrug-2 remained stable in murine liver homogenates        -   c. Prodrug-2 was metabolised rapidly in tumour homogenate            expressing high MT-MMP levels (HT1080) relative to tumour            homogenate expressing low MT-MMP levels (MCF7) (FIG. 4).        -   d. These data support the systemic stability of this prodrug            and the selectivity of activation by MT-MMPs.    -   f) Prodrug-2 is cleaved differentially by MMPs as shown by        LC/MS/MS and mass spectrometry (data not shown):        -   a. Cleaved rapidly at the Glycine-Homophenylalanine            (Gly-Hof) by recombinant MMP-14.        -   b. Cleaved slowly at the Homophenylalanine-Tyrosine            (Hof-Tyr) by recombinant MMP-2. Demonstrating different            cleavage to that observed with prodrug-1.        -   c. Prodrug-2 is not cleaved by recombinant MMP-9, in            contrast to prodrug-1        -   d. These data support the MMP selective cleavage of            prodrug-2    -   g) Prodrug-2 was administered in vivo via the intraperitoneal        route to mice bearing subcutaneous HT1080 tumour model (MT1-MMP        positive). Plasma, tissues and tumours were collected at regular        intervals post-dosing. Levels of prodrug-2 and VDA2 were        assessed by LC/MS/MS.        -   a. Prodrug-2 accumulated and was detected in all tissues            analysed (FIG. 5).        -   b. Highest prodrug-2 levels were observed in the tumour            (FIG. 5).        -   c. Liver was representative of all normal tissues analysed.            (FIG. 5)        -   d. VDA2 was undetectable in plasma following administration            of prodrug-2 (FIG. 6)        -   e. High concentrations of VDA2 were detected in tumour            following prodrug-2 administration (FIG. 6)        -   f. Levels of VDA2 in tumour were 10 times higher than that            detected in normal tissues following administration of            prodrug-2 (FIG. 6)        -   g. High levels of prodrug-2 and VDA2 were still detectable            in tumour 48 hours post administration.

What is claimed is:
 1. A compound or pharmaceutically acceptable saltthereof, comprising a compound X which promotes the formation of stablemicrotubules in a manner similar to paclitaxel associated with a peptideY comprising a matrix metalloproteinase cleavage site comprising theamino acid sequence -Arg-Ser-Cit-Gly-Hof-Tyr-Leu- (SEQ ID NO: 4).
 2. Thecompound or pharmaceutically acceptable salt thereof according to claim1, wherein the compound X is a taxane.
 3. The compound orpharmaceutically acceptable salt thereof according to claim 1, whereinthe compound X is paclitaxel.
 4. The compound or pharmaceuticallyacceptable salt thereof according to claim 1, wherein the compound Xwhich promotes the formation of stable microtubules in a manner similarto paclitaxel is linked directly or indirectly to the peptide.
 5. Thecompound or pharmaceutically acceptable salt thereof according to claim1, further comprising a linker a directly or indirectly associated withX.
 6. The compound or pharmaceutically acceptable salt thereof accordingto claim 5, wherein the linker a is on the C terminus of the amino acidsequence Y.
 7. The compound or pharmaceutically acceptable salt thereofaccording to claim 5 or 6, wherein the linker a may be removedchemically, enzymatically or decompose spontaneously.
 8. The compound orpharmaceutically acceptable salt thereof according to claim 1, furthercomprising a capping group c to prevent non-specific degradation of thepeptide.
 9. The compound or pharmaceutically acceptable salt thereofaccording to claim 8, wherein the capping group c is on the N terminusof the peptide.
 10. The compound or pharmaceutically acceptable saltthereof according to claim 8, wherein the capping group c is selectedfrom simple sugars, D-amino acids, proline imino acids, fluorescein, andfluorescein derivatives.
 11. The compound or pharmaceutically acceptablesalt thereof according to claim 1, further comprising a spacer bdirectly or indirectly associated with the peptide Y.
 12. The compoundor pharmaceutically acceptable salt thereof according to claim 11,wherein the spacer b is on the N terminus of the peptide Y.
 13. Thecompound or pharmaceutically acceptable salt thereof according to claim11, wherein the spacer is selected from a single amino acid, an aminoacid sequence and a succinyl group.
 14. The compound or pharmaceuticallyacceptable salt thereof according to claim 1, wherein the compound hasformula (VI):X-a-Y-b-c  (VI) and each of X, a, Y, b and c are defines as follows: Xis a taxane, a is a linker directly or indirectly associated with X, Yis a peptide comprising a matrix metalloproteinase cleavage sitecomprising the amino acid sequence -Arg-Ser-Cit-Gly-Hof-Tyr-Leu- (SEQ IDNO: 4), b is a spacer directly or indirectly associated with the peptideY, and c is a capping group to prevent non-specific degradation of thepeptide.
 15. A compound or pharmaceutically acceptable salt thereof,comprising paclitaxel conjugated via a linker to a peptide comprising aMMP proteolytic cleavage site and comprising the amino acid sequence-Arg-Ser-Cit-Gly-Hof-Tyr-Leu- (SEQ ID NO: 4).
 16. A pharmaceuticalformulation comprising a compound or pharmaceutically acceptable saltthereof according to claim 1 or 15 and at least one pharmaceuticallyacceptable excipient, diluent or carrier.
 17. A pharmaceuticalformulation according to claim 16, further comprising another anticanceragent.
 18. A method of treating cancer in a subject comprisingadministering to the subject an effective amount of a compound orpharmaceutically acceptable salt thereof according to claim
 15. 19. Amethod of treating cancer in a subject comprising administering to thesubject an effective amount of compound or pharmaceutically acceptablesalt thereof according to claim
 1. 20. The method according to claim 18or 19, wherein the cancer is prostate cancer.
 21. A method of treatingcancer in a subject comprising administering to the subject an effectiveamount of a pharmaceutical formulation comprising a compound accordingto claim
 1. 22. The method according to claim 21, wherein the cancer isprostate cancer.
 23. The compound of claim 10 wherein the fluoresceinderivative is fluorescein isothiocycante (FITC).
 24. The compound ofclaim 13 wherein the single amino acid is β-alanine.